We propose to examine the mechanism(s) of persistence of alphaviruses in tissue culture cells of vertebrates. Three cell systems - murine, hamster, and pig cells persistently infected with the alphavirus Semliki Forest virus will be used in this study. Initially, we shall compare and contrast the biological and physiological status of persistently infected cells in relation to their uninfected progenitors. We shall also compare, in detail, the biological and physical properties of virus particles released from persistently infected cells with those produced during lytic multiplication. This study will involve an examination of the primary amino acid sequence of the structural proteins of the virion and an analysis, by nucleic acid fingerprinting and sequencing, of the viral RNA genome. We shall also examine the molecular biology of virus multiplication in persistently infected cells. In this study, we shall characterize the species of virus-specific RNA isolated from persistently infected cells by affinity chromatography using immobilized viral cDNA made in vitro by avian oncornaviral reverse transcriptase. This characterization will include a study of the messenger potential in vitro of virus-specified mRNA from persistently and lytically infected cells and an examination of the sequence relationship between each virus-specified RNA and the viral genome. As our ongoing sequence studies progress, we should be able to pinpoint any changes which occur in the viral genome which are causally related to the establishment and maintenance of persistence. We shall examine temporal and quantitative aspects of the kinetics of synthesis of virus-specified polypeptides in persistently infected cells. This study will include an examination of both the non-structural (polymerase) and structural polypeptides and a survey of their intracellular site. Finally, an evaluation will be made as to the possible role of viral DNA as a stable repository of viral information in persistently infected cells. Transfection and nucleic acid hybridization will be employed to search for viral DNA in persistently infected cells.